Longevity and Neutralizing Capacity of IgG Antibodies against SARS-CoV-2 Generated by the Application of BNT162b2, AZD1222, Convidecia, Sputnik V, and CoronaVac Vaccines: a Cohort Study in the Mexican Population

ABSTRACT The WHO has approved the use of several vaccines during the COVID-19 pandemic; experience over the last 2 years has indicated that dose demand can only be covered using more than one design. Therefore, having scientific evidence of the performance of the different vaccines applied in a country is highly relevant. In Mexico, 5 vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were used, allowing a cohort study to analyze the generation of anti-S1/S2 IgG antibodies and anti-RBD antibodies with neutralizing activity at 0, 21, 90, and 180 days after vaccination. Five groups of participants were formed on the basis of the type of vaccine received and were divided on the basis of whether they previously had or did not have COVID-19. After completing the vaccination schedule, the seroprevalence was 95.5, 97.5, 81.0, 95.2, and 90.0% (BNT162b2, AZD1222, Convidecia, Sputnik V, and CoronaVac, respectively). Among the participants without COVID-19 prior to vaccination, the largest amount of antibodies in the 90-day period was observed in the BNT162b2 group, and the amount of antibodies in the Sputnik V group decreased the least over time. Even though the percentages of seroconversion obtained in this study were lower than those currently reported in other parts of the world, the tested vaccines are able, in most cases, to induce a good production of IgG antibodies anti-S1/S2 and neutralizing capacity. The fact that there are people who have not produced antibodies during the study leaves open some questions that must be investigated to avoid the appearance of serious cases of COVID-19. IMPORTANCE Since the start of the vaccination programs against COVID-19 in 2020, it was evident that due to global shortages, the demand for the dose required in Mexico could only be covered by acquiring different vaccines. Therefore, determining the effectiveness of these and the longevity of acquired immunity is extremely important in a scenario where SARS-CoV-2 circulation becomes endemic and booster doses are required periodically. Our data reveal significant differences both in the generation of antibodies as well as in their longevity for the vaccines applied in the country but suggest that, in general, the Mexican population can reach a high capacity to neutralize the virus, therefore, regarding less the variant for which they were designed.

know the best vaccine options for the Mexican population. Comments Study is designed well and gives good value to the current scenario. It would be useful and provide more value if this data can be correlated with the presence of a Variant of concerns or variants present during the study time. VOCs do affect the neutralization capacity of vaccine and their efficacy so if it's possible then incorporate or use other published data to describe the possible circuiting VOCs.
Also how you have taken care of the individuals who were infected earlier with COVID and vaccinated or vaccinated and later got infected? Did this impact the outcome?
Did study was followed post-180 days to see the impact of breakthrough, re-infection or other impacts Was booster dose implemented in a later phase How many breakthrough cases were reported during the study Those did not develop antibody, have breakthroughs in follow-up period of 6 months or not Was comorbid group show any significant difference with the healthy group for antibody or NAb difference Was this neutralization enzyme-linked immunosorbent assay (ELISA) specific to measure the neutralizing capacity of the anti-RBD antibodies for any specific VOCs?? Limitation of the study should be expressed Reviewer #2 (Comments for the Author): The work by Fernandes-Matano, Salas-Lais et al. explore the differences at the humoral response level, specifically IgG, in a cohort in Mexico after vaccination with five different vaccines approved in the country against SARS-CoV-2. Several countries in need of controlling SARS-CoV-2 adopted a strategy of vaccinating the population with multiple vaccines based on availability. Therefore, understanding the differences among different vaccine platforms is of interest in other regions and, overall, for pandemic preparedness. The information provided in this article and the samples used for the experiments are valuable. The experimental design is mostly accurate, and the science is sound. However, there are specific flaws in the experimental design mainly associated with ruling out the presence of a natural infection during the experiment that weak the interpretation of the results. The manuscript will considerably benefit if authors decipher whether natural infections happened during the experiment.
Regardless of that, the information is valuable and exciting for the readers of the journals. My specific comments are listed below: Major comments A significant concern is a potential role that natural SARS-CoV-2 infection could have in everyone during the sample collection (Day 0-180). Is the information regarding close contact with SARS-CoV-2-positive people or clinical signs associated with SARS-CoV-2 during the sample collection available? How was the VOC circulation during the experiment timeline? Could this be a variable? Besides CoronaVac, the rest of the vaccines are Spike-based. Therefore, antibodies against N are a tool to differentiate natural infection and vaccination. That information will strongly support the idea of a lack of disease during the sample collection period. Anti-N antibodies are a piece of information already available to the authors based on the discussion.
An increasing body of literature demonstrates that biological sex plays a role in the immune response against vaccination and pathogenesis against respiratory viruses such as influenza or SARS-CoV-2. The authors successfully analyzed potential sex differences, finding such differences in the cohort analyzed (Line 121-23), which is highly informative in this reviewer's opinion. However, the authors failed to show these results appropriately, and just a table is in place. I strongly encourage the authors to present a figure based on sex differences.
Correlations with age were carried out. However, the biological sex was not analyzed.
Please discuss more in detail about potential explanations for the differences in the correlation between S1/S2 IgG vs. neutralization capacity between no prior covid and prior covid positive individuals.  Staff Comments:

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The work by Fernandes-Matano, Salas-Lais et al. explore the differences at the humoral response level, specifically IgG, in a cohort in Mexico after vaccination with five different vaccines approved in the country against SARS-CoV-2. Several countries in need of controlling SARS-CoV-2 adopted a strategy of vaccinating the population with multiple vaccines based on availability. Therefore, understanding the differences among different vaccine platforms is of interest in other regions and, overall, for pandemic preparedness. The information provided in this article and the samples used for the experiments are valuable. The experimental design is mostly accurate, and the science is sound. However, there are specific flaws in the experimental design mainly associated with ruling out the presence of a natural infection during the experiment that weak the interpretation of the results. The manuscript will considerably benefit if authors decipher whether natural infections happened during the experiment.
Regardless of that, the information is valuable and exciting for the readers of the journals. My specific comments are listed below: Major comments A significant concern is a potential role that natural SARS-CoV-2 infection could have in everyone during the sample collection (Day 0-180). Is the information regarding close contact with SARS-CoV-2-positive people or clinical signs associated with SARS-CoV-2 during the sample collection available? How was the VOC circulation during the experiment timeline? Could this be a variable? Besides CoronaVac, the rest of the vaccines are Spike-based. Therefore, antibodies against N are a tool to differentiate natural infection and vaccination. That information will strongly support the idea of a lack of disease during the sample collection period. Anti-N antibodies are a piece of information already available to the authors based on the discussion.
An increasing body of literature demonstrates that biological sex plays a role in the immune response against vaccination and pathogenesis against respiratory viruses such as influenza or SARS-CoV-2. The authors successfully analyzed potential sex differences, finding such differences in the cohort analyzed (Line 121-23), which is highly informative in this reviewer's opinion. However, the authors failed to show these results appropriately, and just a table is in place. I strongly encourage the authors to present a figure based on sex differences.
Correlations with age were carried out. However, the biological sex was not analyzed.
Please discuss more in detail about potential explanations for the differences in the correlation between S1/S2 IgG vs. neutralization capacity between no prior covid and prior covid positive individuals.

Minor comments
Line 61 Response: We included a supplementary figure that shows the VOCs that were circulating during the study period. In addition, we also included a new paragraph on the discussion. Figure S2 shows VOC circulation during the study period, in the area where the samples were obtained. Our data only shows the percentage of neutralizing antibodies vs RBD, but not the neutralizing activity against the different variants. A limitation of our study was not performing neutralization assays against the different variants. Besides, since a large part of the Mexican population was not yet vaccinated during the study period, it is difficult to know if the waves occurred due to the lack of effectiveness of the vaccines used or due to the unvaccinated population.
In a study carried out by Bednarski et al, a reduction in neutralization was observed by Delta and Omicron variants measure by plaque neutralization assays. Even so, although many cases occurred in people vaccinated during the third (delta) and fourth waves (ómicron BA.1 and BA.2), these cases were generally milder and produced significantly fewer deaths, which indicates that even with the reduction of the neutralizing activity, the applied vaccines limited the evolution to severe stages of the disease.
2. Also how you have taken care of the individuals who were infected earlier with COVID and vaccinated or vaccinated and later got infected? Did this impact the outcome?.
Response: Due to the large number of asymptomatic infections, we decided to check whether the participants had already had the disease before vaccination by measuring antibodies against SARS-CoV-2 before immunization with the vaccines studied. Therefore, samples were taken from all the individuals studied minutes before vaccination (day 0), and thus, we were able to identify those who previously had COVID-19 (symptomatic or asymptomatic), from those who had never had contact with the SARS-CoV-2.
Our results show that participants who already had COVID-19 (identified with our assays) generated a much higher number of antibodies compared to the group that did not had COVID before.
Unfortunately, due to lack of resources, we were not able to measure antibodies against N, except for CoronaVac, which would give us information on the participants who had the disease during the study period, therefore, after vaccination. So, we couldn't know what was the impact of getting infected after immunization.
3. Did study was followed post-180 days to see the impact of breakthrough, re-infection or other impacts Response: Yes, we analyzed the participants also at 270 and 360 days post-vaccination, even with the information on the heterologous booster vaccination, however, on the date of submission of this manuscript, we still did not have the analysis of these data and we are preparing for the publication of a second report, in which we will focus the analysis on boosters and heterologous vaccination. Unfortunately, as mentioned in the previous question, it was not possible to identify the subjects who became infected after the start of the study.

Was booster dose implemented in a later phase
Response: Yes, and in most cases the third dose was a different brand of vaccine. This information was not sent, because until the date of sending this manuscript, we still did not have these data, and as we mentioned before, we are still preparing these data for a later report.

How many breakthrough cases were reported during the study
Response: During the study, very few participants reported symptoms similar to those of COVID-19, but we do not have confirmatory data on whether or not they had the disease.
With the intention of verifying if the participants had the infection during the follow-up, we should analyze the different samples in search of antibodies against the N protein (not produced with the vaccine, except for CoronaVac). Nevertheless, because of lack of resources does studies couldn't be done.
6. Those did not develop antibody, have breakthroughs in follow-up period of 6 months or not.
Response: We could not confirm that they had an infection during the study. However, we know that they received one or two doses of the vaccine (depending on the scheme), and that they were also exposed to 3 waves in the country, caused by different variants. Still, there were participants who never developed antibodies, measured by the techniques used in this study.
7. Was comorbid group show any significant difference with the healthy group for antibody or NAb difference Response: Following the reviewer's recommendation, differences in the generation of anti-S1/S2 and RBD antibodies in participants with and without comorbidities were analyzed in more detail. In the group without previous COVID-19, there were no significant differences in terms of the anti-S1/S2 IgG antibody response, nor in the percentage of inhibition in any of the comorbidities analyzed.
However, in the group with previous COVID-19, the people who suffered from arterial hypertension had a higher average of antibodies compared to those who did not suffer from it, a difference that appeared in the first shots, and that was no longer significant. at 180 days of follow-up.
A new paragraph was placed in the discussion section in this regard, and a supplemental table (Supplemental Table 2) was added.
8. Was this neutralization enzyme-linked immunosorbent assay (ELISA) specific to measure the neutralizing capacity of the anti-RBD antibodies for any specific VOCs??
Response: The initial approach aimed to determine the longevity of the antibodies generated by the different vaccines, which were designed based on the wildtype variant, for which ELISA and Neutralization tests designed against the original strain were selected.
However, this does not necessarily mean that they do not detect antibodies produced from infection with other variants.

Limitation of the study should be expressed
Response: The study has 3 important limitations: the first is that for economic reasons it was not possible to determine the presence of antibodies against the N protein, and consequently we could not define infections during follow-up. The second is that plaque reduction neutralization test was not carried out to determine if the neutralizing capacity of the antibodies generated decreases in the face of the different variants, and finally the loss of participants during follow-up.
The paragraph that talks about this in the discussion was edited to make it clearer.
Reviewer 2 1. A significant concern is a potential role that natural SARS-CoV-2 infection could have in everyone during the sample collection (Day 0-180). Is the information regarding close contact with SARS-CoV-2-positive people or clinical signs associated with SARS-CoV-2 during the sample collection available?
Response: We have no information about the close contact of the participants with people with SARS-CoV-2 during the study. The determination of IgG antibodies against the N protein of the virus would have allowed us to better visualize this situation, however, we had economic limitations that did not allow us to do so. Even so, we decided to compare between the groups with and without previous infection, in which the probability of exposition is the same.
2. How was the VOC circulation during the experiment timeline?. Could this be a variable?
Response: We included a supplementary figure 2 that shows the VOCs that were circulating during the study period. In addition we also included a new paragraph on the discussion. Figure S2 shows VOC circulation during the study period, in the area where the samples were obtained. Our data only shows the percentage of neutralizing antibodies vs RBD, but not the neutralizing activity against the different variants. A limitation of our study was not performing neutralization assays against the different variants. Besides, since a large part of the Mexican population was not yet vaccinated during the study period, it is difficult to know if the waves occurred due to the lack of effectiveness of the vaccines used or due to the unvaccinated population.
In a study carried out by Bednarski et al, a reduction in neutralization was observed by Delta and Omicron variants measure by plaque neutralization assays. Even so, although many cases occurred in people vaccinated during the third (delta) and fourth waves (ómicron BA.1 and BA.2), these cases were generally milder and produced significantly fewer deaths, which indicates that even with the reduction of the neutralizing activity, the applied vaccines limited the evolution to severe stages of the disease.
3. Besides CoronaVac, the rest of the vaccines are Spike-based. Therefore, antibodies against N are a tool to differentiate natural infection and vaccination. That information will strongly support the idea of a lack of disease during the sample collection period. Anti-N antibodies are a piece of information already available to the authors based on the discussion.
Response: We agree with the observation, however, we only had access to a small number of reactions against the N protein, so we decided to use them in CoronaVac, since, as we commented in the article, we wanted to verify the data obtained against the S protein in that vaccine in specific, because the individuals immunized with this biological had a different behavior with respect to those who received the other vaccines 4. An increasing body of literature demonstrates that biological sex plays a role in the immune response against vaccination and pathogenesis against respiratory viruses such as influenza or SARS-CoV-2. The authors successfully analyzed potential sex differences, finding such differences in the cohort analyzed (Line 121-23), which is highly informative in this reviewer's opinion. However, the authors failed to show these results appropriately, and just a table is in place. I strongly encourage the authors to present a figure based on sex differences.
Response: In our study, the differences observed between the male and female sex were present only at the first contact with the antigen, so they had only been placed in Table 2.
With the intention of making this result more visible and following the reviewer's recommendation, we have included a new supplementary figure ( Figure S1) 5. Correlations with age were carried out. However, the biological sex was not analyzed.
Response: Comparative analyzes of the amount of anti-S1/S2 IgG antibodies and percentage of inhibition were carried out for both sexes among the participants of the different vaccines, but only sporadic differences were found, which seemed more like random behaviors derived from biological variability, than a real tendency derived from sex.
The only pattern that really appears to be sex-linked is the response generated on first contact with the antigen, which was placed in Table 2, and now also in Supplementary Fig.   1.
6. Please discuss more in detail about potential explanations for the differences in the correlation between S1/S2 IgG vs. neutralization capacity between no prior covid and prior covid positive individuals.
Response: The following paragraph was added to the discussion section The correlation analysis between the amount of IgG produced and the neutralization capacity showed that, contrary to what was expected, these follow a logarithmic relationship and not a linear relationship, which means that, even at the point where a person produces few anti-S1/S2 IgG antibodies, there will not necessarily be a low capacity to neutralize the virus. A clear example that even with a low amount of anti-S1/S2 IgG antibodies, high levels of neutralization can be achieved, is what is observed in the group vaccinated with BNT162b2 after the first immunization. In this group, the logarithmic ratio was lower than the groups vaccinated with other brands, this because more people reached high levels of neutralization, despite the low amount of anti-S1/S2 IgG antibodies they produced in general".
This finding becomes relevant to the interpretation of other works in which population immunity is measured based on kits directed against protein S and not specifically on the ability to neutralize the virus. where a good part of the population has already had a natural SARS-CoV-2 infection, the application of any of the biologicals analyzed in this study manages, in most cases, to induce a good production of anti-S1/S2 and neutralizing IgG antibodies, so they could be an option as a booster dose, as long as take care of the compatibility with the biological applied initially.
The fact that there is a percentage of the population in which there was no production of antibodies during the 180 days of the study leaves open some questions about the effectiveness of the vaccines themselves, the protection generated by the cellular immune response, the form of application, the cold network used to store them and the associated human factor. All these factors must be taken care of and investigated, to avoid the appearance of serious cases of COVID-19.

Minor comments
Line 61-63: Please update the number of cases, deaths, and vaccines administered until September 2022 at least Response: The data was updated until the month of October.
Timeline of sample collection: Please specify specific numbers of individuals and the date of sample collection in materials and methods.
Response: In addition to Figure 9, the number of individuals for the analysis of each vaccine and their sampling date were added in text form.
Fig 1 will benefit from the use of a more precise nomenclature. Along the papers, the different samples are denoted by the day of collection, which I think should be the way to go in this figure.
Response: Correction was made. Response: Because the data in the graphs occupies almost the entire panel, we decided to modify the figure caption to make the information of the days clearer.
Line 161: Please check the figures described. It should be 3C and 3G.
Response: Correction was made. Response: Correction was made. Fig 8 and 9 should be combined.
Response: Correction was made.